help in Plasmid 'mini-prep' experiment - (Jul/04/2006 )
I'm very very confused cos I dont know what is goign on in my lectures adn practicals.. Esp in terms of plasmids adn genes.. Pls could you explain step-by-step what this experiment procedures is use for, eg solution use for what, procedure for what.
Production of Clear Lysate
1. Harvest the over-night bacterial culture (HB101 strain harbouring pGLO plasmid). Centrifuge the cells at 4K rpm for 2 minutes
2. Remove the supernatant and also the excess media by inverting the tube on a paper towel.
3. Add 250 uL of Cell Resuspension Solution (Sol I) and resuspend cell pellet (e.coli) by vortexing. [why do we need to resuspend the cells since supernatant has already been removed? and its in residue already?]
4. Add 250 uL Cell Lysis Solution (Sol II) and mix gently by inverting the tube. DO NOT VORTEX BUT INVERT 4X TO MIX [why??]
5. Incubate until the cell suspension turns slightly clear, approximately 1-5 minutes.
6. Add 10 uL of Alkaline Protease Solution (Sol III) and mix by inverting tube 4 times. Incubate for 5 minutes at room temperature.
7. Add 350 uL of Neutralisation Solution (Sol IV) and immediately mix by inverting tube 4 times. DO NOT VORTEX [why??]
8. Centrifuge the bacterial lysate at 12k rpm for 5 minutes
Binding of Plasmid DNA
9. Insert Spin Column into Collection tube.
10. Decant the clear supernatant/lysate to a Miniprep Spin Column (Avoid transferring any white precipitate)
11. Centrifuge the supernatant at 12k rpm for 1 minute at room temperature.
12. Remove the Spin Column from the tube and discard the flow-through from the Collection Tube.
13. Reinsert the Spin Column into the Collection Tube. Add 750 uL of Column Wash Solution which contains Ethanol (Sol V)
14. Centrifuge at 12k rpm for 1 minute at room temperature
15. Remove the Spin Column from the tube and discard the flow-through from the Collection Tube. Reinsert the Spin Column into the Collection Tube.
16. Repeat wash procedure using 250 uL of Column Wash Solution
17. Centrifuge at 12k rpm for 2 min at room temperature.
18. Discard Flow Tube and transfer the Spin Column to a new, sterile 1.5 mL microfuge tube.
19. Add 50 uL of Nuclease-Free Water (Solution VI) to the Spin Column.
20. Discard Spin Column and keep the plasmid DNA in the microfuge tube. (the extracted plasmid DNA is to be stored at -20 deg c freezer at the end of the expt)
Determination of DNA purity adn Concentration
22. Pippette 20 uL DNA solution into an microfuge tube which contains 480 uL TE buffer.
23. Transfer 500 uL of DNA into a UV cuvette.
24. Read O.D. at 260 nm and 280 nmk to determine the purity ofo the extracted plasmid and also to determine the DNA concentration.
25. Calculate the DNA concentration :
Given: DNA conc = A 260 * 50 * dilution factore
26. Store the remaining DNA in the -20 deg c freezer.
The Qiagen Handbook will help you. I have attached it.
The miniprep protocol starts on page 15 and has many tips & hints as to why each step is taken.
An outline of the principle behind mini preps is on page 8.
If you have anymore questions try to be a little more specific (...in terms of what exact steps you dont understand..... i mean...I am having trouble wording it as I dont want to offend you but.... you have the entire protocol here including "spinning the culture down" and "Inserting the Spin Column into the Collection tube"; you do not understand this? .... I am sure you do so I am hesitant to go to the trouble of explaining each step to you).
As BB would say "Be precise" and I am more than happy to help...surely there are more steps harder to understand than others.
Also, in the next post perhaps you could suggest reasons why you think they are done.
For step 3, why do we need to resuspend the cells since supernatant has already been removed? and its in residue already?
[3. Add 250 uL of Cell Resuspension Solution (Sol I) and resuspend cell pellet (e.coli) by vortexing.]
The Cell Lysis Solution (Sol II) is to lyse the cells to release the plasmids and DNA? but why cannot vortex teh tube?
For step 6, what is the sue of the Alkaline Protease Solution (Sol III)? Is it to remove the proteins from the plasmid to get a purer plasmid yield?
Step 7, what is the Neutralisation Solution (Sol IV) for? to neutralise what? Cos there's no acid or alkali inside, right?
Is the washing done to purify the plasmid DNA?
In step 19, what is the Nuclease-Free Water (Solution VI) for? I dont quite know what nuclease is in the first place.
Sorry for not specifying the questions earlier on. I hope you can help me to clarify my doubts. thanks!
You resuspend the cells because you need not to have clumps when you lyse the cells and you cannot vortex when you add the lysis buffer.
You cannot vortex when you add the lysis solution because you don't want to shear the DNA.
The alkaline solution contains SDS and NaOH, to denature proteins and RNA.
The neutralization solution will lower the pH to 8, when the genomic DNA, the proteins and the RNA will precipitate while the plasmid DNA will stay in solution.
The washing is to remove whatever you wouldn't like to have together with your DNA.
The nuclease is any enzyme that can digest the DNA.
It doesn't want to be a bad comment but it's just a curiosity: didn't you have a course atthe uni where they explained this things to you? I had to study it, that's why I'm asking...
nope. they juz gave experiment procedures in the lab manual and told me to follow through. So.. it's like, for me, "huh??" cos I dont know what I'm doing. my yield was a low 13.6% only. hahaha!!
thanks for your help!!
the reason you have to pellet and resuspend the cells and you can't just lyse them in the juice they are already in, is that it's spent media...too salty, dirty with cellular waste and dead cell bits...you need the cells to be 'clean' before you crack them open